USPTO Patents Application 10551504

Applicant : Hiroyuki Tsunoda et al. Attorney's Docket No.: 14875-0153US1 / C1-A0320Y2P-US

Serial No. : 10/551,504

Filed : May 12, 2006

Page : 2 of 14

Amendments to the Claims :
This listing of claims replaces all prior versions and listings of claims in the application:

Listing of Claims :

1. (Original) An antibody comprising a single-chain polypeptide having binding activity
against TPO receptor (Mpl), wherein said antibody comprises two heavy chain variable regions
and two light chain variable regions.

2. (Original) The antibody of claim 1, wherein the two heavy chain variable regions and
the two light chain variable regions are arranged in the order of heavy chain variable region, light
chain variable region, heavy chain variable region, and light chain variable region from the N
terminus of the single-chain polypeptide.

3. (Previously Presented) The antibody of claim 1, wherein the two heavy chain variable
regions and the two light chain variable regions are linked by linkers.

4. (Original) The antibody of claim 3, wherein the linkers comprise 15 amino acids.

5. (Original) A chimeric antibody that binds to Mpl.

6. (Original) The antibody of claim 5, which is a humanized antibody.

7. (Previously Presented) The antibody of claim 5, which is a minibody.

8. (Original) An antibody that binds to soluble Mpl.

Applicant : Hiroyuki Tsunoda et al. Attorney's Docket No.: 14875-0153US1 / C1-A0320Y2P-US

Serial No. : 10/551,504

Filed : May 12, 2006

Page : 3 of 14

9. (Withdrawn) An antibody that binds to human Mpl and monkey Mpl.

10. (Withdrawn) An antibody having agonistic activity against human Mpl and monkey

Mpl.

11. (Original) An antibody whose binding activity to soluble Mpl is KD = 10" 6 M or

lower.

12. (Original) An antibody whose binding activity to soluble Mpl is KD = 10" 7 M or

lower.

13. (Original) An antibody whose TPO agonistic activity is EC50 = 100 nM or lower.

14. (Original) An antibody whose TPO agonistic activity is EC50 = 30 nM or lower.

15. (Original) An antibody whose TPO agonistic activity is EC50 = 10 nM or lower.

16. (Currently Amended) The antibody of claim 32 that comprises a heavy chain
variable region, wherein said heavy chain variable region comprises CDR1, CDR2 and CDR3
consisting of an amino acid sequence of any one of:

f±4 (a) SEQ ID NOs: 3, 4, and 5
{24 (b) SEQ ID NOs: 6, 7, and 8
f34(c)SEQIDNOs: 9, 10, and 11
{4} (d) SEQ ID NOs: 15, 16, and 17
f5j (e) SEQ ID NOs: 18, 19, and 20
f64 (0 SEQ ID NOs: 2 1, 22, and 23
P} (g) SEQ ID NOs: 24, 25, and 26

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Attorney's Docket No.: 14875-0153US1 / C1-A0320Y2P-US

£8} (h) SEQ ID NOs: 27, 28, and 29
£9} (0 SEQ ID NOs: 30, 3 1 , and 32
£40} (j) SEQ ID NOs: 33, 34, and 35
£444 (k) SEQ ID NOs: 36, 37, and 38
£424 0} SEQ ID NOs: 39, 40, and 41
£454 (m) SEQ ID NOs: 42, 43, and 44
£44} (n) SEQ ID NOs: 48, 49, and 50
£454 (o) SEQ ID NOs: 51, 52, and 53
M£e> SEQ ID NOs: 54, 55, and 56
£±3} (a) SEQ ID NOs: 57, 58, and 59.

17. (Currently Amended) The antibody of claim 32 that comprises a light chain variable
region, wherein said light chain variable region comprises CDR1, CDR2 and CDR3 consisting of
an amino acid sequence of any one of:

£4} (a) SEQ ID NOs: 60, 61, and 62

£24 (b) SEQ ID NOs: 63, 64, and 65

£34 (c) SEQ ID NOs: 78, 79, and 80

£4}{d} SEQ ID NOs: 84, 85, and 86

£5} (e) SEQ ID NOs: 93, 94, and 95

£64 (0 SEQ ID NOs: 96, 97, and 98

£7} (g) SEQ ID NOs: 102, 103, and 104

£8}(h) SEQ ID NOs: 108, 109, and 110

£9} (i) SEQ ID NOs: 111, 112, and 113

£40] 0} SEQ ID NOs: 114, 115, and 116.

18. (Currently Amended) The antibody of claim 32 that comprises a heavy chain
variable region and a light chain variable region of any one of:

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f4j(aj a heavy chain variable region that comprises CDR1, CDR2, and CDR3 comprising
the amino acid sequences consisting of SEQ ID NOs: 3, 4, and 5, and a light chain variable
region that comprises CDR1, CDR2, and CDR3 comprising the amino acid sequences consisting
of SEQ ID NOs: 60, 61, and 62;

£24, (b) a heavy chain variable region that comprises CDR1, CDR2, and CDR3
comprising the amino acid sequences consisting of SEQ ID NOs: 6, 7, and 8, and a light chain
variable region that comprises CDR1, CDR2, and CDR3 comprising the amino acid sequences
consisting of SEQ ID NOs: 63, 64, and 65;

f3j(c) a heavy chain variable region that comprises CDR1, CDR2, and CDR3 comprising
the amino acid sequences consisting of SEQ ID NOs: 9, 10, and 11, and a light chain variable
region that comprises CDR1, CDR2, and CDR3 comprising the amino acid sequences consisting
of SEQ ID NOs: 63, 64, and 65;

f4}(d) a heavy chain variable region that comprises CDR1, CDR2, and CDR3
comprising the amino acid sequences consisting of SEQ ID NOs: 15, 16, and 1 7, and a light
chain variable region that comprises CDR1, CDR2, and CDR3 comprising the amino acid
sequences consisting of SEQ ID NOs: 63, 64, and 65;

f#}(e) a heavy chain variable region that comprises CDR1, CDR2, and CDR3 comprising
the amino acid sequences consisting of SEQ ED NOs: 18, 19, and 20, and a light chain variable
region that comprises CDR1, CDR2, and CDR3 comprising the amino acid sequences consisting
of SEQ ED NOs: 63, 64, and 65;

{6}{fJ a heavy chain variable region that comprises CDR1, CDR2, and CDR3 comprising
the amino acid sequences consisting of SEQ ID NOs: 21, 22, and 23, and a light chain variable
region that comprises CDR1, CDR2, and CDR3 comprising the amino acid sequences consisting
of SEQ ID NOs: 78, 79, and 80;

f7} (g) a heavy chain variable region that comprises CDR1, CDR2, and CDR3
comprising the amino acid sequences consisting of SEQ ID NOs: 24, 25, and 26, and a light
chain variable region that comprises CDR1, CDR2, and CDR3 comprising the amino acid
sequences consisting of SEQ ID NOs: 63, 64, and 65;

Applicant : Hiroyuki Tsunoda et al. Attorney's Docket No.: 14875-0153US1 / C1-A0320Y2P-US

Serial No. : 10/551,504
Filed : May 12, 2006
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£8} (h) a heavy chain variable region that comprises CDR1 , CDR2, and CDR3
comprising the amino acid sequences consisting of SEQ ID NOs: 27, 28, and 29, and a light
chain variable region that comprises CDR1, CDR2, and CDR3 comprising the amino acid
sequences consisting of SEQ ID NOs: 84, 85, and 86;

£9} (0 a heavy chain variable region that comprises CDR1, CDR2, and CDR3 comprising
the amino acid sequences consisting of SEQ ID NOs: 30, 3 1 , and 32, and a light chain variable
region that comprises CDR1, CDR2, and CDR3 comprising the amino acid sequence consisting
of SEQ ID NOs: 63, 64, and 65;

£fO](j} a heavy chain variable region that comprises CDR1, CDR2, and CDR3
comprising the amino acid sequences consisting of SEQ ID NOs: 33, 34, and 35, and a light
chain variable region that comprises CDR1, CDR2, and CDR3 comprising the amino acid
sequences consisting of SEQ ID NOs: 63, 64, and 65;

£444, (k) a heavy chain variable region that comprises CDR1, CDR2, and CDR3
comprising the amino acid sequences consisting of SEQ ID NOs: 36, 37, and 38, and a light
chain variable region that comprises CDR1, CDR2, and CDR3 comprising the amino acid
sequences consisting of SEQ ID NOs: 93, 94, and 95;

£424, (1) a heavy chain variable region that comprises CDR1, CDR2, and CDR3
comprising the amino acid sequences consisting of SEQ ID NOs: 39, 40, and 41, and a light
chain variable region that comprises CDR1, CDR2, and CDR3 comprising the amino acid
sequences consisting of SEQ ED NOs: 96, 97, and 98;

£434, {m} a heavy chain variable region that comprises CDR1, CDR2, and CDR3
comprising the amino acid sequences consisting of SEQ ID NOs: 42, 43, and 44, and a light
chain variable region that comprises CDR1, CDR2, and CDR3 comprising the amino acid
sequences consisting of SEQ ID NOs: 78, 79, and 80;

{44} (n) a heavy chain variable region that comprises CDR1, CDR2, and CDR3
comprising the amino acid sequences consisting of SEQ ID NOs: 45, 46, and 47, and a light
chain variable region that comprises CDR1, CDR2, and CDR3 comprising the amino acid
sequences consisting of SEQ ID NOs: 102, 103, and 104;

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£4#] (o) a heavy chain variable region that comprises CDR1, CDR2, and CDR3
comprising the amino acid sequences consisting of SEQ ID NOs: 48, 49, and 50, and a light
chain variable region that comprises CDR1, CDR2, and CDR3 comprising the amino acid
sequences consisting of SEQ ID NOs: 63, 64, and 65;

f46} (p) a heavy chain variable region that comprises CDR1, CDR2, and CDR3
comprising the amino acid sequences consisting of SEQ ID NOs: 51, 52, and 53, and a light
chain variable region that comprises CDR1, CDR2, and CDR3 comprising the amino acid
sequences consisting of SEQ ID NOs: 108, 109, and 110,

(474 (q) a heavy chain variable region that comprises CDR1, CDR2, and CDR3
comprising the amino acid sequences consisting of SEQ ID NOs: 54, 55, and 56, and a light
chain variable region that comprises CDR1, CDR2, and CDR3 comprising the amino acid
sequences consisting of SEQ ID NOs: 111, 112, and 113;

{4-84, (r) a heavy chain variable region that comprises CDR1, CDR2, and CDR3
comprising the amino acid sequences consisting of SEQ ID NOs: 57, 58, and 59, and a light
chain variable region that comprises CDR1, CDR2, and CDR3 each comprising the amino acid
sequences consisting of SEQ ID NOs: 114, 115, and 1 16.

19. (Previously Presented) The antibody of claim 32 that comprises a heavy chain
variable region comprising the amino acid sequence of SEQ ID NO: 118.

20. (Previously Presented) The antibody of claim 32 that comprises a light chain
variable region comprising the amino acid sequence of SEQ ID NO: 120.

21. (Previously Presented) The antibody of claim 32 that comprises a heavy chain
variable region comprising the amino acid sequence of SEQ ID NO: 1 18 and a light chain
variable region comprising the amino acid sequence of SEQ ID NO: 120.

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22. (Previously Presented) The antibody of claim 32 that comprises the amino acid
sequence of SEQ ID NO: 122 or 264.

23. (Currently Amended) The antibody of claim 16, wherein said heavy chain variable
region comprises FR1, FR2, FR3, and FR4 consisting of amino acid sequences of any one of

f44-(a) SEQ ID NOs: 230, 232, 234, and 236
{24(b) SEQ ID NOs: 265, 267, 269, and 271
f3}£s) SEQ ID NOs: 279, 281, 283, and 285
f4} (d) SEQ ID NOs: 298, 299, 300, and 301
{5} (e) SEQ ED NOs: 298, 299, 306, and 301 .

24. (Currently Amended) The antibody of claim 17, wherein said light chain variable
region comprises FR1, FR2, FR3, and FR4 consisting of amino acid sequences of any one of:

f4}(a) SEQ ID NOs: 239, 241, 243, and 245
f2}(b) SEQ ID NOs: 272, 274, 276, and 278
f3}(c) SEQ ID NOs: 302, 303, 304, and 305
f4} (d) SEQ ID NOs: 302, 307, 308, and 305.

25. (Currently Amended) The antibody of claim 18 that comprises a heavy chain
variable region and a light chain variable region of any one of:

J44, (a) a heavy chain variable region which comprises FR1, FR2, FR3, and FR4 having
the amino acid sequences consisting of SEQ ID NOs: 230, 232, 234, and 236, and a light chain
variable region which comprises FR1, FR2, FR3, and FR4 having the amino acid sequences
consisting of SEQ ED NOs: 239, 241, 243, and 245;

f2} (b) a heavy chain variable region which comprises FR1, FR2, FR3, and FR4 having
the amino acid sequences consisting of SEQ ID NOs: 265, 267, 269, and 271, and a light chain
variable region which comprises FR1, FR2, FR3, and FR4 having the amino acid sequences
consisting of SEQ ID NOs: 272, 274, 276, and 278;

Applicant : Hiroyuki Tsunoda et al. Attorney's Docket No.: 14875-0153US1 / C1-A0320Y2P-US

Serial No. : 10/551,504

Filed : May 12, 2006

Page : 9 of 14

{3}{c) a heavy chain variable region which comprises FR1, FR2, FR3, and FR4 having
the amino acid sequences consisting of SEQ ID NOs: 279, 281, 283, and 285, and a light chain
variable region which comprises FR1, FR2, FR3, and FR4 having the amino acid sequences
consisting of SEQ ID NOs: 272, 274, 276, and 278;

f44 (d) a heavy chain variable region which comprises FR1, FR2, FR3, and FR4 having
the amino acid sequences consisting of SEQ ID NOs: 298, 299, 300, and 301, and a light chain
variable region which comprises FR1, FR2, FR3, and FR4 having the amino acid sequences
consisting of SEQ ID NOs: 302, 303, 304, and 305;

£5}£e) a heavy chain variable region which comprises FR1, FR2, FR3, and FR4 having
the amino acid sequences consisting of SEQ ID NOs: 298, 299, 306, and 301, and a light chain
variable region which comprises FR1, FR2, FR3, and FR4 having the amino acid sequences
consisting of SEQ ID NOs: 302, 307, 308, and 305.

26. (Previously Presented) The antibody of claim 32 that comprises a heavy chain
variable region, wherein said heavy chain variable region comprises the amino acid sequence of
SEQ ID NO: 229, 256, 262, 289, or 295.

27. (Previously Presented) The antibody of claim 32 that comprises a light chain
variable region, wherein said light chain variable region comprises the amino acid sequence of
SEQ ID NO: 238, 258, 291, or 297.

28. (Currently Amended) The antibody of claim 32 that comprises a heavy chain
variable region and a light chain variable region of any one of:

{44 £a) a heavy chain variable region comprising the amino acid sequence of SEQ ID NO:
229, and a light chain variable region comprising the amino acid sequence of SEQ ID NO: 238;

{2} (b) a heavy chain variable region comprising the amino acid sequence of SEQ ID NO:
256, and a light chain variable region comprising the amino acid sequence of SEQ ID NO: 258;

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{3} (c) a heavy chain variable region comprising the amino acid sequence of SEQ ID NO:
262, and a light chain variable region comprising the amino acid sequence of SEQ ID NO: 258;

{4} (d) a heavy chain variable region comprising the amino acid sequence of SEQ ID NO:
289, and a light chain variable region comprising the amino acid sequence of SEQ ID NO: 291;

(e) a heavy chain variable region comprising the amino acid sequence of SEQ ID NO:
295, and a light chain variable region comprising the amino acid sequence of SEQ ID NO: 297.

29. (Previously Presented) The antibody of claim 32 that comprises the amino acid
sequence of SEQ ID NO: 2, 254, 260, 287, or 293.

30. -31. (Canceled)

32. (Original) An antibody that recognizes the region of amino acids 26 to 274 of human

Mpl.

33. (Previously Presented) An antibody of claim 1, which has TPO agonistic activity.

34. (Withdrawn) A polynucleotide encoding an antibody of claim 1.

35. (Withdrawn) A polynucleotide hybridizing to the polynucleotide of claim 34 under
stringent conditions, wherein said polynucleotide encodes an antibody having binding activity
against TPO receptor (Mpl).

36. (Withdrawn) A vector comprising the polynucleotide of claim 34.

37. (Withdrawn) A host cell that carries the polynucleotide of claim 34.

Applicant : Hiroyuki Tsunoda et al. Attorney's Docket No.: 14875-0 1 53US 1 / CI -A0320Y2P-US

Serial No. : 10/551.504

Filed : May 12, 2006

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38. (Previously Presented) A pharmaceutical composition comprising an antibody of
claim 32.

39. (Withdrawn) The antibody of claim 32, wherein the antibody recognizes an epitope
recognized by an antibody consisting of the amino acid sequence of SEQ ID NO:2.

40. (Withdrawn) The antibody of claim 32, wherein the antibody recognizes an epitope
within the region of amino acids 189 to 245 of human Mpl.

41. (Withdrawn) The antibody of claim 32, wherein the antibody recognizes an epitope
within the region of amino acids 213 to 231 of human Mpl.

42. (Previously Presented) The antibody of claim 32, wherein the antibody comprises a
heavy chain variable region containing a CDR1 amino acid sequence of SEQ ID NO:36 in which
one or more amino acid residues has been substituted, deleted, added, and/or inserted; a CDR2
amino acid sequence of SEQ ID NO:37 in which one or more amino acid residues has been
substituted, deleted, added, and/or inserted; and a CDR3 amino acid sequence of SEQ ID NO:38
in which one or more amino acid residues has been substituted, deleted, added, and/or inserted,
wherein the antibody has an activity of a second antibody whose heavy chain variable region
comprises SEQ ED NOs:36, 37 and 38 and whose light chain variable region SEQ ID NOs:93, 94
and 95.

43. (Previously Presented) The antibody of claim 42, wherein the antibody binds to the
same epitope as the second antibody.